DESCRIPTION: The long-term objectives of this project are to understand and control butyrate and pentanoate metabolism in streptomycetes. In particular, the role of butyrate and pentanoate building blocks in processes of polyketide and fatty acid biosynthesis will be studied. The long-term benefits of this research may include methods for: 1) production of novel antibiotics with improved therapeutic activity, 2) increased yields of natural products from fermentation processes, and 3) genetic manipulation of strains so that they produce a single natural product, rather than a mixture of related natural products. The central theme of the research is a mechanistic, stereochemical, enzymatic and genetic investigation of the following three enzymes: crotonyl CoA reductase (CCR), isobutyryl CoA mutase (ICM), and beta-ketoacyl synthase III (KASIII). These enzymes are thought to catalyze critical steps in three separate pathways for providing butyrate building blocks in streptomycetes: A hypothesis that all three pathways can function to produce butyrate building blocks for straight-chain fatty acid biosynthesis, but that only the pathways using CCR and ICM function to provide butyrate building blocks for polyketide biosynthesis, will be tested. This work will be accomplished by a series of gene disruption experiments in Streptomyces collinus and Streptomyces cinnamonensis (producer of the polyketide antibiotic monensin A which contains a butyrate-derived moiety). Studies of enoyl thioester reductase FabI, putatively involved in a KASIII catalyzed pathway for butyrate formation with also be undertaken. The methods used for determining the role of this enzyme will involve enzyme purification and characterization, gene cloning and sequencing, and functional analysis by gene disruption. Finally the pathways for formation of the unusual pentanoate/pentenoate building block, used in the biosynthesis of the polyketide immunosuppressant FK506, will be studied. A comparison with the pathways of formation of butyrate building blocks will be made.